Bacillus amyloliquefaciens fcc1256 compositions and methods of controlling plant pathogens

ABSTRACT

The present application discloses method of controlling plant pathogen(s), e.g. fungal and bacterial pathogens, on a plant, wherein a composition comprising  Bacillus amyloliquefaciens  FCC1256 deposited as ATCC No. PTA-122162 is applied to the plant, in particular to over-ground parts of the plant. The composition may comprise iturin and fengycin in a relative weight ratio of 1.3:1.0 to 3.0:1.0. The application also discloses an agricultural composition comprising the novel strain, a carrier, a surface-active agent and optionally a buffer, and a corresponding concentrate.

TECHNICAL FIELD

The presently disclosed subject matter relates to compositionscomprising an isolated Bacillus amyloliquefaciens FCC1256 strain forapplication to plants or the soil surrounding plants to treat plantdiseases caused by plant pathogens.

BACKGROUND

Fungal plant pathogens (phytopathogens), including but not limited toBotrytis spp., Fusarium spp., Rhizoctonia spp. are one type of plantpest that can cause severe economic losses in the agricultural andhorticultural industries. Chemical agents can be used to control fungalphytopathogens, but the use of chemical agents suffers fromdisadvantages including high cost, lack of efficacy, emergence ofresistant strains of the fungi, and undesirable environmental impacts.In addition, such chemical treatments tend to be indiscriminant and mayadversely affect beneficial bacteria, fungi, and arthropods in additionto the plant pathogen at which the treatments are targeted. A secondtype of plant pest are bacterial plant pathogens, including but notlimited to Erwinia spp., Xanthomonas spp., and Pseudomonas spp. thatcause severe economic losses in the agricultural and horticulturalindustries. Similar to pathogenic fungi, the use of chemical agents totreat these bacterial pathogens suffers from disadvantages. Thus,microorganisms that can be applied as biopesticides to controlpathogenic fungi and bacteria in plants are desirable and in high demandto improve agricultural sustainability.

Some members of the species Bacillus have been reported as biocontrolstrains, and some have been applied in commercial products. For example,strains currently being used in commercial biocontrol products include:Bacillus velezensis QST713, used as the active ingredient of Serenade®,produced by Bayer Crop Science, and Bacillus amyloliquefaciens D747,used as the active ingredient in Double Nickel produced by Certis. Inaddition, Bacillus strains currently being used in commercialbiostimulant products include: Bacillus velezensis FZB42 used as theactive ingredient in RhizoVital® 42, produced by ABiTEP GmbH.

Moreover, WO 2016/109396 A1 discloses Bacillus amyloliquefaciens RTI472for use in benefiting plant growth and for treating plant diseases likefungal and bacterial infections.

Further, WO 2016/109395 A1 discloses Bacillus amyloliquefaciens RTI301for use in benefitting plant growth and for treating plant diseases likefungal and bacterial infections.

Also, WO 2015/023662 A1 discloses, i.a., a Bacillus amyloliquefaciensstrain for inhibiting the growth and/or activity of fungal plantpathogens.

However, despite the availability of certain biological pesticides,there is a need in the art to improve specificity against the targetdiseases and their respective causal pathogens. Further, there is a needto improve efficacy and potency of biological pesticides which may beimpacted by various biotic and abiotic factors. The present inventionaddresses the need in the art by providing novel microbial compositionsand methods for their use to control the growth of plant pathogens andthereby alleviate or prevent plant diseases.

SUMMARY OF THE INVENTION

The present invention resides in the isolation of a new Bacillusamyloliquefaciens strain from strawberry plant soil. The strain ofBacillus amyloliquefaciens FCC1256 has been deposited at the ATCC underPatent Accession No. PTA-122162, cf. Example 1.

In at least one embodiment, the present invention is directed to methodsof controlling plant pathogen(s) on a plant, the methods comprising thestep of applying a composition comprising Bacillus amyloliquefaciensFCC1256 to the plant, to a part of the plant and/or to a locus at whichthe plant or plant part grows or is to be planted.

In another embodiment, the present invention relates to a method ofcontrolling fungal plant pathogen(s) and/or bacterial plant pathogen(s)on a plant, the method comprising the step of applying a compositioncomprising Bacillus amyloliquefaciens FCC1256 to over-ground parts ofthe plant.

In one embodiment, an agricultural composition is provided comprising a)Bacillus amyloliquefaciens FCC1256, and b) an agriculturally suitableformulation medium, e.g. comprising a suitable carrier, a surface-activeagent, and optionally a buffer. The agricultural composition isparticularly useful for application to protect plants against pathogensor treat a pathogenic infection in a susceptible plant, e.g. accordingto the methods described herein. In some embodiments, the compositioncomprises iturin and fengycin in a relative ratio of in the range offrom 1.3:1.0 to 3.0:1.0, such as from 1.5:1.0 to 2.8:1.0, respectively.

In further embodiments, a concentrate for an agricultural composition isprovided, the concentrate comprising: a) Bacillus amyloliquefaciensFCC1256, and b) an agriculturally suitable formulation medium, e.g. amedium comprising a suitable carrier, a surface-active agent, andoptionally a buffer.

BRIEF DESCRIPTION OF THE FIGURES

Having thus described the presently disclosed subject matter in generalterms, reference will now be made to the accompanying Figures describedin the following:

FIG. 1 shows the results of the Detached Pepper Leaf Disk assaycomparing the activity of Bacillus amyloliquefaciens FCC1256 tountreated controls as well as to the commercial product Serenade®Optimum.

FIG. 2 shows the typical ratios of iturin, surfactin, fengycin andericin A/S from FCC1256 and RTI472 cultures, respectively.

DETAILED DESCRIPTION

As used herein, the designations “Bacillus amyloliquefaciens FCC1256”and

“FCC1256” refer to a biologically pure culture of the Bacillusamyloliquefaciens FCC1256 bacterial strain deposited under PatentAccession No. PTA-122162, cf. Example 1. In the methods, compositions,etc. described herein, the strain may be present in one or a combinationof forms: as isolated spores of the bacterial stain, as a fermentationbroth comprising spores of the bacterial strain, as a processedfermentation product comprising spores of the bacterial strain, asisolated vegetative cells of the bacterial strain, as a fermentationbroth comprising vegetative cells of the bacterial strain, and as aprocessed fermentation product comprising vegetative cells of thebacterial strain.

The term “biologically pure culture” refers a laboratory or fermentationculture which contains a single species of organism, i.e. in the presentcase virtually exclusively a Bacillus amyloliquefaciens FCC1256bacterial strain. This means that any other microorganisms involved inthe fermentation broth of the pure culture are considered ascontaminations, which only exist in a negligible amount and does notcause measurable changes to the physical and chemical compositions ofthe broth.

The term “processed fermentation product” refers to a downstreamprocessed form thereof, including but not limited to a concentrate ofthe fermentation broth, solids of a filtered fermentation broth, areconstituted fermentation broth concentrate, and a dried fermentationbroth (e.g. freeze-dried or spray-dried).

In some embodiments, FCC1256 is present as a fermentation brothcomprising spores of FCC1256 or a processed fermentation productcomprising spores of FCC1256, such as a concentrate of a fermentationbroth or a spray-dried fermentation broth.

The fact that a biologically pure culture of Bacillus amyloliquefaciensFCC1256 is included in the compositions and methods disclosed hereindoes not exclude that the compositions and methods, respectively mayinclude other defined microbial strains, in particular otherbiologically pure microbial strains.

The terms “a,” “an,” and “the” refer to “one or more” when used in thisapplication, including the claims. Thus, for example, reference to “aplant” includes a plurality of plants, unless the context clearly is tothe contrary.

Throughout this specification and the claims, the terms “comprise,”“comprises,” and “comprising” are used in a non-exclusive sense, exceptwhere the context requires otherwise. Likewise, the term “include” andits grammatical variants are intended to be non-limiting, such thatrecitation of items in a list is not to the exclusion of other likeitems that can be substituted or added to the listed items.

For the purposes of this specification and claims, the term “about” whenused in connection with one or more numbers or numerical ranges, shouldbe understood to refer to all such numbers, including all numbers in arange and modifies that range by extending the boundaries above andbelow the numerical values set forth. The recitation of numerical rangesby endpoints includes all numbers, e.g., whole integers, includingfractions thereof, subsumed within that range (for example, therecitation of 1 to 5 includes 1, 2, 3, 4, and 5, as well as fractionsthereof, e.g., 1.5, 2.25, 3.75, 4.1, and the like) and any range withinthat range.

For the purposes of this specification and claims, the terms“metabolite” and “metabolites” are used in connection with compoundshaving antimicrobial activity that are produced by Bacillusamyloliquefaciens FCC1256, such as iturin, fengycin and surfactin. Themetabolites referred to as “iturin”, “fengycin” and “surfactin” eachrepresent a class of rather similar molecular structures. In so far thatreference is made to amounts or ratios of including iturin, fengycin andsurfactin, the amount/ratio is intended to relate to the total amount(weight) of the respective metabolite. The amount can be determined byHPLC analysis using suitable commercial standards.

Methods of the Invention

In certain embodiments of the present invention, compositions andmethods are provided that include Bacillus amyloliquefaciens FCC1256 forapplication to a plant for conferring protection against a pathogenicinfection in a susceptible plant. By the methods of the presentinvention, the controlling a plant pathogen may be reflected in reducedpathogenic infection, improved resistance to plant pathogens (e.g.resistance to subsequent attacks), improved seedling vigor, improvedroot development, improved plant growth, improved plant health,increased yield, improved appearance, or a combination thereof.

Hence, the present invention i.a. relates to a method of controllingplant pathogen(s) on a plant, the method comprising the step of applyinga composition comprising Bacillus amyloliquefaciens FCC1256 to theplant, to a part of the plant and/or to a locus at which the plant orplant part grows or is to be planted.

As used herein, the term “plant pathogen” refers to organisms that causeinfectious disease in plants, including fungi, bacteria, viruses,viroids, virus-like organisms, oomycetes, phytoplasmas, protozoa,nematodes and parasitic plants. In some embodiments, plant pathogens areselected from fungi and bacteria. In one variant, plant pathogens areselected from fungal plant pathogens. In another variant, plantpathogens are selected from bacterial plant pathogens.

In another embodiment, the method includes applying the compositioncomprising Bacillus amyloliquefaciens FCC1256 to the plant, to a part ofthe plant and/or to a locus at which the plant or plant part grows or isto be planted to control plant pathogens selected from the groupconsisting of a rust fungus, a Botrytis spp. (like Botrytis cinerea,Botrytis squamosal), an Erwinia spp. (like Erwinia carotovora, Erwiniaamylovora), a Dickeya spp. (like Dickeya dadantii, Dickeya solani), anAgrobacterium spp. (like Agrobacterium tumefaciens, Agrobacteriumtumefaciens), a Xanthomonas spp. (like Xanthomonas axonopodis,Xanthomonas campestris pv. carotae, Xanthomonas pruni, Xanthomonasarboricola, Xanthomonas oryzae pv. oryzae), a Xylella spp. (like Xylellafastidiosa), a Candidatus spp. (like Candidatus liberibacter), aFusarium spp. (like Fusarium colmorum, Fusarium graminearum, Fusariumoxysporum, Fusarium oxysporum f. sp. cubense, Fusarium oxysporum f. sp.lycopersici, Fusarium virguliforme), a Sclerotinia spp. (likeSclerotinia sclerotiorum, Sclerotinia minor, Sclerotinia homeocarpa), aCercospora/Cercosporidium spp., an Uncinula spp. (like Uncinulanecator), a Podosphaera spp. (like Podosphaera leucotricha, Podosphaeraclandestine), a Phomopsis spp. (like Phomopsis viticola), an Alternariaspp. (like Alternaria tenuissima, Alternaria porri, Alternariaalternate, Alternaria solani, Alternaria tenuis), a Pseudomonas spp.(like Pseudomonas syringae pv. Tomato), a Phytophthora spp. (likePhytophthora infestans, Phytophthora parasitica, Phytophthora sojae,Phytophthora capsici, Phytophthora cinnamon, Phytophthora fragariae,Phytophthora ramorum, Phytophthora palmivara, Phytophthora nicotianae),a Phakopsora spp. (like Phakopsora pachyrhizi, Phakopsora meibomiae), anAspergillus spp. (like Aspergillus flavus, Aspergillus niger), aUromyces spp. (like Uromyces appendiculatusi), a Cladosporium spp. (likeCladosporium herbarum), a Rhizopus spp. (like Rhizopus arrhizus), aRhizoctonia spp. (like Rhizoctonia solani, Rhizoctonia zeae, Rhizoctoniaoryzae, Rhizoctonia caritae, Rhizoctonia cerealis, Rhizoctonia crocorum,Rhizoctonia fragariae, Rhizoctonia ramicola, Rhizoctonia rubi,Rhizoctonia leguminicola), a Macrophomina spp. (like Macrophominaphaseolina), a Magnaporthe spp. (like Magnaporthe grisea, Magnaportheoryzae), a Mycosphaerella spp. (like Mycosphaerella graminocola,Mycosphaerella fijiensis (Black sigatoga), Mycosphaerella pomi,Mycosphaerella citri), a Monilinia spp. (like Monilinia fruticola,Monilinia vacciniicorymbosi, Monilinia laxa), a Colletotrichum spp.(like Colletotrichum gloeosporiodes, Colletotrichum acutatum,Colletotrichum Candidum), a Diaporthe (spp. Diaporthe citri), aCorynespora spp. (like Corynespora Cassiicola), a Gymnosporangium spp.(like Gymnosporangium jumperi-virginianae), a Schizothyrium spp. (likeSchizothyrium pomi), a Gloeodes spp. (like Gloeodes pomigena), aBotryosphaeria spp. (like Botryosphaeria dothidea), a Neofabraea spp., aWilsonomyces spp. (like Wilsonomyces carpophilus), a Sphaerotheca spp.(like Sphaerotheca macularis, Sphaerotheca pannosa), an Erysiphe spp., aStagonospora spp. (like Stagonospora nodorum), a Pythium spp. (likePythium ultimum, Pythium aphanidermatum, Pythium irregularum, Pythiumulosum, Pythium lutriarium, Pythium sylvatium), a Venturia spp. (likeVenturia inaequalis), a Verticillium spp., a Ustilago spp. (likeUstilago nuda, Ustilago maydis, Ustilago scitaminea), a Claviceps spp.(like Claviceps puprrea), a Tilletia spp. (like Tilletia tritici,Tilletia laevis, Tilletia horrid, Tilletia controversa), a Phoma spp.(like Phoma glycinicola, Phoma exigua, Phoma lingam), a Cocliobolus spp.(like Cocliobolus sativus), a Gaeumanomyces spp. (Gaeumanomycesgaminis), a Colleototricum spp., a Rhychosporium spp. (likeRhychosporium secalis), a Biopolaris spp., a Helminthosporium spp. (likeHelminthosporium secalis, Helminthosporium maydis, Helminthosporiumsolai, Helminthosporium tritici-repentis), and a combinations thereof,including any subspecies variants thereof.

In some embodiments, the method is adapted to control one or more plantpathogens selected from Botrytis cinerea, Botrytis squaomas, Fusariumgraminearum, Fusarium oxysporum, Fusarium viguliforme, Phytophthorainfestans, Phytophthora parasitica, Phytophthora sojae, Phytophthoracapsici, Phytophthora cinnamon, Phytophthora fragariae, Phytophthoraramorum, Phytophthora palmivara, Phytophthora nicotianae, Sclerotiniasclerotiorum, Sclerotinia minor, Sclerotinia homeocarpa, Aspergillusfavus, Pseudomonas syringae pv. Tomato, Erwinia amylovara, Rhizoctoniasolani, Xanthomonas euvesicatoria, and any combinations thereof,including any subspecies variants thereof.

In some embodiments, the methods of the invention utilize a compositioncomprising Bacillus amyloliquefaciens FCC1256 to controls diseasescaused by such plant pathogens as pepper Grey Mold (Botrytis cinerea),pepper Phytophthora Blight (Phytophthora capsica), Tomato Fusarium crownrot (Fusarium sp.), Apple Fire Blight (Erwinia amylovora), TomatoBacterial Speck (Pseudomonas syringae), Pepper Bacterial Spot disease(Xanthomonas euvesicatoria), soybean damping off (Rhizoctonia solani).

As used herein, the phrase “controlling plant pathogen(s)” in thecontext of the methods and compositions of the present invention isintended to mean an at least 10% reduction of the growth of the plantpathogen(s) compared to corresponding conditions where the methods orthe compositions are not utilized, e.g. in some embodiments an at least30% reduction, or an at least 50% reduction, or an at least 70%reduction, or an at least 80% reduction, or an at least 90% reduction,or essentially an elimination of the growth. In the latter instance, theelimination of the growth may result in visual elimination of the plantpathogen(s). Examples 3, 5, 6 and 7 demonstrate types of methodologiesfor determination of the degree of control of various plant pathogens byutilizing Bacillus amyloliquefaciens FCC1256.

The methods and compositions according to the invention are useful toprevent or treat diseases caused by plant pathogens in a broad range ofplant including, but not limited to, Corn, Sweet Corn, Seed Corn, SilageCorn, Field Corn, Rice, Wheat, Barley, Sorghum, Asparagus, Blueberry,Blackberry, Raspberry, Loganberry, Huckleberry, Cranberry, Gooseberry,Elderberry, Currant, Cane berry, Bush berry, Broccoli, Cabbage,Cauliflower, Brussels Sprouts, Collards, Kale, Mustard Greens, Kohlrabi,Cucumber, Cantaloupe, Melon, Muskmelon, Squash, Watermelon, Pumpkin,Eggplant, Onion, Garlic, Shallots, Orange, Grapefruit, Lemon, Tangerine,Tangelo, Pomelo, Pepper, Tomato, Ground Cherry, Tomatillo, Okra, Grape,Lettuce, Celery, Spinach, Parsley, Radicchio, Beans, Green beans, Snapbeans, Shell beans, Soybeans, Dry Beans, Garbanzo beans, Lima beans,Peas, Chick peas, Split peas, Lentils, Canola, Castor, Coconut, Cotton,Flax, Oil Palm, Olive, Peanut, Rapeseed, Safflower, Sesame, Sunflower,Soybean, Apple, Crabapple, Pear, Quince, May haw, Carrot, Potato, SweetPotato, Cassava, Beets, Ginger, Horseradish, Radish, Ginseng, Turnip,Apricot, Cherry, Nectarine, Peach, Plum, Prune, Strawberry, Almond,Pistachio, Pecan, Walnut, Filberts, Chestnut, Cashew, Beechnut,Butternut, Macadamia, Kiwi, Banana, (Blue) Agave, Grass, Turf grass,Poinsettia, Chestnuts, Oak, Maple, sugarcane, and sugar beet.

In some embodiments, the plant is soybean, bean, snap bean, wheat,cotton, corn, pepper, tomato, potato, cassava, grape, strawberry,banana, peanut, squash, pumpkin, eggplant, and cucumber.

In other embodiments, the plant is selected from apples, asparagus,bayberry, bearberry, blackberry, highbush and lowbush blue berry,chokecherry, currant, elderberry, gooseberry, huckleberry, lingonberry,loganberry, mulberry, pin cherry, raspberry, Salaberry, Saskatoon berry,sea buckthorn and wild raspberry, blackberry or raspberry, grapes,strawberry, fuzzy kiwifruit, broccoli, Chinese broccoli, broccoli raab,Brussel sprouts, cabbage, cauliflower, broccoli, collard, kale,kohlrabi, mizuna, mustard greens, mustard spinach and rape greens,onions, garlic, leeks, shallot and chives, celery, cantaloupe, Chinesewas gourd, cucumber, edible gourd, melon, muskmelon, pumpkin, squash,watermelon, eggplant, ground cherry, okra, peppers, tomatillos andtomato.

In still other embodiments, the plant is selected from pepper,cucumbers, apples, asparagus, bananas, citrus, kiwi, melons, peaches,pears, pineapple, pome fruit, pomegranate, celery, onions, garlic,grapes, leaks, shallots, chives, broccoli, cabbage, cauliflower,cucurbits, tomatoes, potatoes, wheat, rice or soybeans.

In at least one embodiment of the methods of the invention, thecomposition is applied to the plant, to a part of the plant and/or to alocus at which the plant or plant part grows or is to be planted, e.g.foliage of the plant, bark of the plant, fruit of the plant, flowers ofthe plant, seed of the plant, roots of the plant, a cutting of theplant, a graft of the plant, soil or growth medium surrounding theplant; soil or growth medium before sowing seeds of the plant in thesoil or growth medium; or soil or growth medium before planting theplant, the plant cutting, or the plant graft in the soil or growthmedium.

In one variant, the invention relates to a method of controlling fungalplant pathogen(s) and/or bacterial plant pathogen(s) on a plant, themethod comprising applying a composition comprising Bacillusamyloliquefaciens FCC1256 deposited as ATCC No. PTA-122162 toover-ground parts of the plant.

As used herein, the phrase “applying [ . . . ] to over-ground parts of aplant”, and the like, refers to application of the composition to theplant by aiming at the stem(s), the leave(s), the flower(s) and/or thefruit(s) of the plant. In other embodiments, application is aimed at thelocus at which the plant or plant parts grows or is to be planted, e.g.to root system of the plant or the soil around the roots system of theplant, or to the soil in which the plant (or plant parts) is to beplanted; this collectively being referred to as “applying [ . . . ] tothe soil around the plant”.

Applying a composition comprising Bacillus amyloliquefaciens FCC1256 tothe plant, to a part of the plant and/or to a locus at which the plantor plant part grows or is to be planted may be accomplished by anyconventional means, e.g. by spray application, by dripping, by powdering(powder application), by application of a foam (foam application), etc.

For applications to over-ground parts of a plant, spray application orpowder application may prove particularly interesting.

For applications to the soil around the plant, spray application,application by dripping, powder application and foam application may beinteresting. In one variant, application to the soil around a plant isby in-furrow application by dripping or foam application in connectionwith seed planting.

In some embodiments, the pathogenic infection can be caused by one or acombination of:

Soybean rust fungi (Phakopsora pachyrhizi, Phakopsora meibomiae) and theplant is soybean;

Grey Mold (Botrytis cinerea) and the plant is grape, strawberry orpepper;

Alternaria spp. (e.g. Alternaria solani) and the plant is tomato orpotato;

Bean Rust (Uromyces appendiculatus) and the plant is common bean;

Microsphaera diffusa (Soybean Powdery Mildew) and the plant is soybean;

Mycosphaerella fijiensis (Black sigatoga) or Fusarium oxysporum f. sp.cubese (Panama disease) and the plant is banana;

Xanthomonas spp. or Xanthomonas oryzae pv. oryzae and the plant is rice;

Xanthomonas axonopodis and the plant is cassava;

Xanthomonas campestris and the plant is tomato;

Powdery mildew and the plant is a cucurbit;

Southern White Mold and the plant comprises peanut;

Leaf spot (Cercospora/Cercosporidium) and the plant is peanut;

Fusarium graminearum (Wheat Head Scab) and the plant is wheat;

Mycosphaerella graminicola (Septoria tritici blotch) and the plant iswheat;

Stagonospora nodorum (glume blotch and Septoria nodorum blotch), and theplant is wheat;

Erwinia amylovora, and the plant is selected from apple, pear and otherpome fruits;

Venturia inaequalis, and the plant is selected from apple, pear andother pome fruits;

Sclerotinia sclerotiorum (white mold) and the plant is snap bean orpotato;

Sclerotinia homeocarpa (dollar spot) and the plant comprises turf grass;or

Rhizoctonia solani and the plant is selected from wheat, rice, turfgrass, soybean, corn, legumes and vegetable crops.

In one variant of the above-mentioned embodiment, the plant pathogen(s)is(are) controlled by application of the composition to the over-groundparts of the plant.

In embodiments herein, the composition is applied in an effectiveamount. The term “effective amount” refers to an amount of thecomposition which is sufficient to control at least one plant pathogen.

In embodiments, the composition is applied such that the rate ofBacillus amyloliquefaciens FCC1256 is in the range of from 4.0×10⁹CFU/ha to 4.0×10¹⁷ CFU/ha, such as 4.0×10¹⁰ CFU/ha to 4.0×10¹⁶ CFU/ha.Typically, Bacillus amyloliquefaciens FCC1256 is applied in the form ofspores thereof, rather than vegetative cells.

In some embodiments of the method of the invention, the composition isin liquid form, and the Bacillus amyloliquefaciens FCC1256 is present inthe composition at a concentration of from 1.0×10⁶ CFU/mL to 1.0×10¹²CFU/mL, such as from 1.0×10⁷ CFU/mL to 1.0×10¹¹ CFU/mL.

In embodiments where the applied composition is in liquid form, thecomposition may be in the form of a liquid formulation selected fromsuspensions, suspension concentrates (SC), oil dispersions (OD) andfoams.

In other embodiments of the method of the invention, the composition isin solid form, and the Bacillus amyloliquefaciens FCC1256 is present inthe composition at a concentration of from 1.0×10⁶ CFU/g to 1.0×10¹²CFU/g, such as from 1.0×10⁷ CFU/g to 1.0×10¹¹ CFU/g.

In embodiments where the applied composition is in solid form, thecomposition may be in the form of a formulation selected from dustablepowders (DP), water-dispersible granules (WG) and wettable powders (WP).

In some embodiments, the applied composition comprises iturin andfengycin in a relative weight ratio of in the range of from 1.3:1.0 to3.0:1.0, such as from 1.5:1.0 to 2.8:1.0 and all measurements withinsuch ranges.

The nature, embodiments and variants of the composition comprisingBacillus amyloliquefaciens FCC1256 utilized in the method of theinvention will be further described in the following under the heading“Agricultural compositions of the invention”.

Agricultural Compositions of the Invention

The composition as described herein and the embodiments thereof furtherelaborated on in the following are particularly useful in the methoddescribed hereinabove, but are not limited to such application. Thecompositions described under the present heading are intended to beready-to-use compositions. In some embodiments, it may be convenient toprepare concentrated compositions suited for dilution before use toarrive at the ready-to-use agricultural compositions, e.g. to reducetransportation costs or to prolong the shelf-life, etc. Suchconcentrated formulations are described further below under the heading“Concentrates for agricultural compositions”.

Hence, the present invention i.a. relates to an agricultural compositioncomprising: a) Bacillus amyloliquefaciens, and b) an agriculturallysuitable formulation medium.

The term “agriculturally suitable” is intended to mean that theformulation medium as such (i.e. without any deliberate inclusion offurther active ingredients) should not have significant detrimentaleffects on the plant or plants to which the agricultural composition isintended to be applied. Accordingly, “agriculturally suitableformulation medium” include any such formulation medium in whichBacillus amyloliquefaciens FCC1256 can be placed in to facilitatetransport of an effective amount to be applied to the plant part ofinterest, and which is otherwise suitable for agricultural use.

The formulation medium may include a liquid or solid carrier, as well asone or more additional components selected from surface-active agents,preservatives, humectants, desiccants, anti-foam agents, anti-freezeagents, dispersants, binders, emulsifiers, dyes, ultra-violet lightprotectants, drift-control agents, spray deposition aids, free-flowagents, buffers, and thickeners, and combinations thereof.

Examples of liquid carriers include water, animal oils and derivatives,mineral oils and derivatives, vegetable oils and derivatives, alcohols,polyols, triglycerides, natural and synthetic polymers and nonionicderivatives thereof. Examples of solid carriers include minerals, clays,silicas, inorganic and organic salts, sugars, starch, waxes, groundanimal shells, botanical material including fibers, husks shells andflour.

In embodiments, the formulation medium comprises a suitable carrier anda surface-active agent. In variants hereof, the formulation mediumfurther comprises a buffer.

In some embodiment, the agricultural composition comprises iturin andfengycin in a relative weight ratio of in the range of from 1.3:1.0 to3.0:1.0, such as from 1.5:1.0 to 2.8:1.0, including all measurementswithin such ranges.

In some embodiments, the composition may be in the form of a formulationselected from suspensions, suspension concentrates (SC), oil dispersions(OD), foams, dustable powders (DP), water-dispersible granules (WG) andwettable powders (WP).

Typically, Bacillus amyloliquefaciens FCC1256 is included in suitableformulations in the form of spores thereof, rather than in the form ofvegetative cells. For example, the formulations may include an aliquotof a fermentation broth comprising spores of FCC1256 or a processedfermentation product comprising spores of FCC1256, such as a concentrateof a fermentation broth or a spray-dried fermentation broth.

In some embodiments, the agricultural composition is in liquid form, andthe Bacillus amyloliquefaciens FCC1256 is present in the composition ata concentration of from 1.0×10⁶ CFU/mL to 1.0×10¹² CFU/mL, such as from1.0×10⁷ CFU/mL to 1.0×10¹¹ CFU/mL.

In embodiments where the agricultural composition is in liquid form, thecomposition may be in the form of a formulation selected fromsuspensions, suspension concentrates (SC), oil dispersions (OD) andfoams.

In other embodiments, the agricultural composition is in solid form, andthe Bacillus amyloliquefaciens FCC1256 is present in the composition ata concentration of from 1.0×10⁶ CFU/g to 1.0×10¹² CFU/g, such as from1.0×10⁷ CFU/g to 1.0×10¹¹ CFU/g.

In embodiments where the agricultural composition is in solid form, thecomposition may be in the form of a formulation selected from dustablepowders (DP), water-dispersible granules (WG) and wettable powders (WP).

In the methods for delivering FCC1256 in combination with a microbial, abiological, or a chemical insecticide, fungicide, nematicide,bacteriocide, or plant growth regulator, the composition can be in theform of a liquid, an oil dispersion, a dust, a dry wettable powder, aspreadable granule, or a dry wettable granule.

In some embodiments, the agricultural compositions may, in addition tothe formulation medium, further include one or a combination of amicrobial or a chemical insecticide, fungicide, nematicide,bacteriocide, plant growth regulator, or plant growth promotor, e.g.:

Insecticides: A0) various insecticides, including agrigata,al-phosphide, amblyseius, aphelinus, aphidius, aphidoletes, artimisinin,autographa californica NPV, azocyclotin, Bacillus subtilis, Bacillusthuringiensis spp. aizawai, Bacillus thuringiensis spp. kurstaki,Bacillus thuringiensis, Beauveria bassiana, beta-cyfluthrin, bisultap,brofluthrinate, bromophos-e, bromopropylate, capsaicin, cartap,celastrus-extract, chlorbenzuron, chlorethoxyfos, chlorfluazuron,cnidiadin, cryolite, cyanophos, cyhalothrin, cyhexatin, cypermethrin,dacnusa, DCIP, dichloropropene, dicofol, diglyphus, diglyphus+dacnusa,dimethacarb, emamectin, encarsia, EPN, eretmocerus, ethylene-dibromide,eucalyptol, fenazaquin, fenobucarb (BPMC), fenpyroximate,flubrocythrinate, flufenzine, formetanate, formothion, furathiocarb,gamma-cyhalothrin, garlic-juice, granulosis-virus, harmonia, heliothisarmigera NPV, indol-3-ylbutyric acid, iodomethane, iron, isocarbofos,isofenphos, isofenphos-m, isoprocarb, isothioate, lindane, liuyangmycin,matrine, mephosfolan, metaldehyde, metarhizium-anisopliae,methamidophos, metolcarb (MTMC), mirex, m-isothiocyanate, monosultap,myrothecium verrucaria, naled, neochrysocharis formosa, nicotine,nicotinoids, omethoate, orius, oxymatrine, paecilomyces, parathion-e,pasteuria, pheromones, phosphorus-acid, photorhabdus, phoxim,phytoseiulus, pirimiphos-e, Plutella xylostella GV, polyhedrosis-virus,polyphenol-extracts, potassium-oleate, profenofos, prosuler, prothiofos,pyraclofos, pyrethrins, pyridaphenthion, pyrimidifen, pyriproxifen,quillay-extract, quinomethionate, rotenone, saponin, saponozit,sodium-fluosilicate, ssulfluramid, sulphur, tebupirimfos, tefluthrin,temephos, tetradifon, thiofanox, thiometon, transgenics (e.g., Cry3Bb1),triazamate, trichoderma, trichogramma, triflumuron, verticillium,vertrine, kappa-bifenthrin, kappa-tefluthrin, dichoromezotiaz,broflanilide, pyraziflumid; A1) the class of carbamates, includingaldicarb, alanycarb, benfuracarb, carbaryl, carbofuran, carbosulfan,methiocarb, methomyl, oxamyl, pirimicarb, propoxur and thiodicarb; A2)the class of organophosphates, including acephate, azinphos-ethyl,azinphos-methyl, chlorfenvinphos, chlorpyrifos, chlorpyrifos-methyl,demeton-S-methyl, diazinon, dichlorvos/DDVP, dicrotophos, dimethoate,disulfoton, ethion, fenitrothion, fenthion, isoxathion, malathion,methamidaphos, methidathion, mevinphos, monocrotophos, oxymethoate,oxydemeton-methyl, parathion, parathion-methyl, phenthoate, phorate,phosalone, phosmet, phosphamidon, pirimiphos-methyl, quinalphos,terbufos, tetrachlorvinphos, triazophos and trichlorfon; A3) the classof cyclodiene organochlorine compounds such as endosulfan; A4) the classof fiproles, including ethiprole, fipronil, pyrafluprole and pyriprole;A5) the class of neonicotinoids, including acetamiprid, clothianidin,dinotefuran, imidacloprid, nitenpyram, thiacloprid and thiamethoxam; A6)the class of spinosyns such as spinosad and spinetoram; A7) chloridechannel activators from the class of mectins, including abamectin,emamectin benzoate, ivermectin, lepimectin and milbemectin; A8) juvenilehormone mimics such as hydroprene, kinoprene, methoprene, fenoxycarb andpyriproxyfen; A9) selective homopteran feeding blockers such aspymetrozine, flonicamid and pyrifluquinazon; A10) mite growth inhibitorssuch as clofentezine, hexythiazox and etoxazole; A11) inhibitors ofmitochondrial ATP synthase such as diafenthiuron, fenbutatin oxide andpropargite; uncouplers of oxidative phosphorylation such aschlorfenapyr; A12) nicotinic acetylcholine receptor channel blockerssuch as bensultap, cartap hydrochloride, thiocyclam and thiosultapsodium; A13) inhibitors of the chitin biosynthesis type 0 from thebenzoylurea class, including bistrifluron, diflubenzuron, flufenoxuron,hexaflumuron, lufenuron, novaluron and teflubenzuron; A14) inhibitors ofthe chitin biosynthesis type 1 such as buprofezin; A15) moultingdisruptors such as cyromazine; A16) ecdyson receptor agonists such asmethoxyfenozide, tebufenozide, halofenozide and chromafenozide; A17)octopamin receptor agonists such as amitraz; A18) mitochondrial complexelectron transport inhibitors pyridaben, tebufenpyrad, tolfenpyrad,flufenerim, cyenopyrafen, cyflumetofen, hydramethylnon, acequinocyl orfluacrypyrim;A19) voltage-dependent sodium channel blockers such asindoxacarb and metaflumizone; A20) inhibitors of the lipid synthesissuch as spirodiclofen, spiromesifen and spirotetramat; A21) ryanodinereceptor-modulators from the class of diamides, including flubendiamide,the phthalamide compounds(R)-3-Chlor-N1-{2-methyl-4-[1,2,2,2-tetrafluor-1-(trifluormethyl)ethyl]phenyl}-N2-(1-methyl-2-methylsulfonylethyl)phthalamidand(S)-3-Chlor-N1-{2-methyl-4[1,2,2,2-tetrafluor-1-(trifluormethyl)ethyl]phenyl}-N2-(1-methyl-2-methylsulfonylethyl)phthalamid,chlorantraniliprole and cyantraniliprole; A22) compounds of unknown oruncertain mode of action such as azadirachtin, amidoflumet, bifenazate,fluensulfone, piperonyl butoxide, pyridalyl, sulfoxaflor; or A23) sodiumchannel modulators from the class of pyrethroids, including acrinathrin,allethrin, bifenthrin, cyfluthrin, lambda-cyhalothrin, cypermethrin,alpha-cypermethrin, beta-cypermethrin, zeta-cypermethrin, deltamethrin,esfenvalerate, etofenprox, fenpropathrin, fenvalerate, flucythrinate,tau-fluvalinate, permethrin, silafluofen and tralomethrin.

Fungicides/bacteriocides: B0) benzovindiflupyr, anitiperonosporic,ametoctradin, amisulbrom, copper salts (e.g., copper hydroxide, copperoxychloride, copper sulfate, copper persulfate), boscalid, thiflumazide,flutianil, furalaxyl, thiabendazole, benodanil, mepronil, isofetamid,fenfuram, bixafen, fluxapyroxad, penflufen, sedaxane, coumoxystrobin,enoxastrobin, flufenoxystrobin, pyraoxystrobin, pyrametostrobin,triclopyricarb, fenaminstrobin, metominostrobin, pyribencarb,meptyldinocap, fentin acetate, fentin chloride, fentin hydroxide,oxytetracycline, chlozolinate, chloroneb, tecnazene, etridiazole,iodocarb, prothiocarb, Bacillus subtilis, extract from Melaleucaalternifolia, extract from Lupinus albus doce, BLAD polypeptide,pyrisoxazole, oxpoconazole, etaconazole, fenpyrazamine, naftifine,terbinafine, validamycin, pyrimorph, valifenalate, fthalide,probenazole, isotianil, laminarin, extract from Reynoutriasachalinensis, phosphorous acid and salts, teclofthalam, triazoxide,pyriofenone, organic oils, potassium bicarbonate, chlorothalonil,fluoroimide; B1) azoles, including bitertanol, bromuconazole,cyproconazole, difenoconazole, diniconazole, enilconazole,epoxiconazole, fluquinconazole, fenbuconazole, flusilazole, flutriafol,hexaconazole, imibenconazole, ipconazole, metconazole, myclobutanil,penconazole, propiconazole, prothioconazole, simeconazole, triadimefon,triadimenol, tebuconazole, tetraconazole, triticonazole, prochloraz,pefurazoate, imazalil, triflumizole, cyazofamid, benomyl, carbendazim,thiabendazole, fuberidazole, ethaboxam, etridiazole and hymexazole,azaconazole, diniconazole-M, oxpoconazol, paclobutrazol, uniconazol,1-(4-chloro-phenyl)-2-([1,2,4]triazol-1-yl)-cycloheptanol andimazalilsulfphate; B2) strobilurins, including azoxystrobin,dimoxystrobin, enestroburin, fluoxastrobin, kresoxim-methyl,methominostrobin, orysastrobin, picoxystrobin, pyraclostrobin,trifloxystrobin, enestroburin, methyl(2-chloro-5-[1-(3-methylbenzyloxyimino)ethyl]benzyl)carbamate, methyl(2-chloro-5-[1-(6-methylpyridin-2-ylmethoxyimino)ethyl]benzyl)carbamateand methyl2-(ortho-(2,5-dimethylphenyloxymethylene)-phenyl)-3-methoxyacrylate,2-(2-(6-(3-chloro-2-methyl-phenoxy)-5-fluoro-pyrimidin-4-yloxy)-phenyl)-2-methoxyimino-N-methyl-acetamideand3-methoxy-2-(2-(N-(4-methoxy-phenyl)-cyclopropanecarboximidoylsulfanylmethyl)-phenyl)-acrylicacid methyl ester; B3) carboxamides, including carboxin, benalaxyl,benalaxyl-M, fenhexamid, flutolanil, furametpyr, mepronil, metalaxyl,mefenoxam, ofurace, oxadixyl, oxycarboxin, penthiopyrad, isopyrazam,thifluzamide, tiadinil,3,4-dichloro-N-(2-cyanophenyl)isothiazole-5-carboxamide, dimethomorph,flumorph, flumetover, fluopicolide (picobenzamid), zoxamide,carpropamid, diclocymet, mandipropamid,N-(2-(4-[3-(4-chlorophenyl)prop-2-ynyloxy]-3-methoxyphenyl)ethyl)-2-methanesulfonyl-amino-3-methylbutyramide,N-(2-(4-[3-(4-chloro-phenyl)prop-2-ynyloxy]-3-methoxy-phenyl)ethyl)-2-ethanesulfonylamino-3-methylbutyramide,methyl3-(4-chlorophenyl)-3-(2-isopropoxycarbonyl-amino-3-methyl-butyrylamino)propionate,N-(4′-bromobiphenyl-2-yl)-4-difluoromethyl-methylthiazole-δ-carboxamide,N-(4′-trifluoromethyl-biphenyl-2-yl)-4-difluoromethyl-2-methylthiazole-5-carboxamide,N-(4′-chloro-3′-fluorobiphenyl-2-yl)-4-difluoromethyl-2-methyl-thiazole-5-carboxamide,N-(3\4′-dichloro-4-fluorobiphenyl-2-yl)-3-difluoro-methyl-1-methyl-pyrazole-4-carboxamide,N-(3′,4′-dichloro-5-fluorobiphenyl-2-yl)-3-difluoromethyl-1-methylpyrazole-4-carboxamide,N-(2-cyano-phenyl)-3,4-dichloroisothiazole-5-carboxamide,2-amino-4-methyl-thiazole-5-carboxanilide,2-chloro-N-(1,1,3-trimethyl-indan-4-yl)-nicotinamide,N-(2-(1,3-dimethylbutyl)-phenyl)-1,3-dimethyl-5-fluoro-1H-pyrazole-4-carboxamide,N-(4′-chloro-3′,5-difluoro-biphenyl-2-yl)-3-difluoromethyl-1-methyl-1H-pyrazole-4-carboxamide,N-(4′-chloro-3′,5-difluoro-biphenyl-2-yl)-3-trifluoromethyl-1-methyl-1H-pyrazole-4-carboxamide,N-(3′,4′-dichloro-5-fluoro-biphenyl-2-yl)-3-trifluoromethyl-1-methyl-1H-pyrazole-4-carboxamide,N-(3′,5-difluoro-4′-methyl-biphenyl-2-yl)-3-difluoromethyl-1-methyl-1H-pyrazole-4-carboxamide,N-(3′,5-difluoro-4′-methyl-biphenyl-2-yl)-3-trifluoromethyl-1-methyl-1H-pyrazole-4-carboxamide,N-(cis-2-bicyclopropyl-2-yl-phenyl)-3-difluoromethyl-1-methyl-1H-pyrazole-4-carboxamide,N-(trans-2-bicyclopropyl-2-yl-phenyl)-3-difluoro-methyl-1-methyl-1H-pyrazole-4-carboxamide,fluopyram,N-(3-ethyl-3,5-5-trimethyl-cyclohexyl)-3-formylamino-2-hydroxy-benzamide,oxytetracyclin, silthiofam, N-(6-methoxy-pyridin-3-yl)cyclopropane-carboxamide, 2-iodo-N-phenyl-benzamide,N-(2-bicyclo-propyl-2-yl-phenyl)-3-difluormethyl-1-methylpyrazol-4-ylcarboxamide;B4) heterocyclic compounds, including fluazinam, pyrifenox, bupirimate,cyprodinil, fenarimol, ferimzone, mepanipyrim, nuarimol, pyrimethanil,triforine, fenpiclonil, fludioxonil, aldimorph, dodemorph,fenpropimorph, tridemorph, fenpropidin, iprodione, procymidone,vinclozolin, famoxadone, fenamidone, octhilinone, probenazole,5-chloro-7-(4-methyl-piperidin-1-yl)-6-(2,4,6-trifluorophenyl)-[1,2,4]triazolo[1,5-a]pyrimidine,anilazine, diclomezine, pyroquilon, proquinazid, tricyclazole,2-butoxy-6-iodo-3-propylchromen-4-one, acibenzolar-S-methyl, captafol,captan, dazomet, folpet, fenoxanil, quinoxyfen,N,N-dimethyl-3-(3-bromo-6-fluoro-2-methylindole-1-sulfonyl)-[1,2,4]triazole-1-sulfonamide,5-ethyl-6-octyl-[1,2,4]triazolo[1,5-a]pyrimidin-2,7-diamine,2,3,5,6-tetrachloro-4-methanesulfonyl-pyridine,3,4,5-trichloro-pyridine-2,6-di-carbonitrile,N-(1-(5-bromo-3-chloro-pyridin-2-yl)-ethyl)-2,4-dichloro-nicotinamide,N-((5-bromo-3-chloropyridin-2-yl)-methyl)-2,4-dichloro-nicotinamide,diflumetorim, nitrapyrin, dodemorphacetate, fluoroimid, blasticidin-S,chinomethionat, debacarb, difenzoquat, difenzoquat-methylsulphat,oxolinic acid and piperalin; B5) carbamates, including mancozeb, maneb,metam, methasulphocarb, metiram, ferbam, propineb, thiram, zineb, ziram,diethofencarb, iprovalicarb, benthiavalicarb, propamocarb, propamocarbhydrochlorid, 4-fluorophenylN-(1-(1-(4-cyanophenyl)-ethanesulfonyl)but-2-yl)carbamate, methyl3-(4-chloro-phenyl)-3-(2-isopropoxycarbonylamino-3-methyl-butyrylamino)propanoate;or B6) other fungicides, including guanidine, dodine, dodine free base,iminoctadine, guazatine, kasugamycin, oxytetracyclin and its salts,streptomycin, polyoxin, validamycin A, binapacryl, dinocap, dinobuton,dithianon, isoprothiolane, fentin salts, edifenphos, iprobenfos,fosetyl, fosetyl-aluminum, phosphorous acid and its salts, pyrazophos,tolclofos-methyl, dichlofluanid, flusulfamide, hexachloro-benzene,phthalide, pencycuron, quintozene, thiophanate, thiophanate-methyl,tolylfluanid, cyflufenamid, cymoxanil, dimethirimol, ethirimol,furalaxyl, metrafenone and spiroxamine, guazatine-acetate,iminoctadine-triacetate, iminoctadine-tris(albesilate), kasugamycinhydrochloride hydrate, dichlorophen, pentachlorophenol and its salts,N-(4-chloro-2-nitro-phenyl)-N-ethyl-4-methyl-b enzenesulfonamide,dicloran, nitrothal-isopropyl, tecnazen, biphenyl, bronopol,diphenylamine, mildiomycin, oxincopper, prohexadione calcium,N-(cyclopropylmethoxy-imino-(6-difluoromethoxy-2,3-difluoro-phenyl)-methyl)-2-phenylacetamide,N′-(4-(4-chloro-3-trifluoromethyl-phenoxy)-2,5-dimethyl-phenyl)-N-ethyl-N-methylformamidine,N′-(4-(4-fluoro-3-trifluoromethyl-phenoxy)-2,5-dimethyl-phenyl)-N-ethyl-N-methylformamidine,N′-(2-methyl-5-trifluormethyl-4-(3-trimethylsilanyl-propoxy)-phenyl)-N-ethyl-N-methylformamidine,N′-(5-difluormethyl-2-methyl-4-(3-trimethylsilanyl-propoxy)-phenyl)-N-ethyl-N-methylformamidine, and fluindapyr.

Nematicides: C1) Benomyl, cloethocarb, aldoxycarb, tirpate, diamidafos,fenamiphos, cadusafos, dichlofenthion, ethoprophos, fensulfothion,fosthiazate, heterophos, isamidofof, isazofos, phosphocarb, thionazin,imicyafos, mecarphon, acetoprole, benclothiaz, chloropicrin, dazomet,fluensulfone, 1,3-dichloropropene (telone), dimethyl disulfide, metamsodium, metam potassium, metam salt (all MITC generators), methylbromide, biological soil amendments (e.g., mustard seeds, mustard seedextracts), steam fumigation of soil, allyl isothiocyanate (AITC),dimethyl sulfate, furfual (aldehyde).

Plant growth regulators: D1) Antiauxins, such as clofibric acid,2,3,5-tri-iodobenzoic acid; D2) Auxins such as 4-CPA, 2,4-D, 2,4-DB,2,4-DEP, dichlorprop, fenoprop, IAA, IBA, naphthaleneacetamide,a-naphthaleneacetic acids, 1-naphthol, naphthoxyacetic acids, potassiumnaphthenate, sodium naphthenate, 2,4,5-T; D3) cytokinins, such as 2iP,benzyladenine, 4-hydroxyphenethyl alcohol, kinetin, zeatin; D4)defoliants, such as calcium cyanamide, dimethipin, endothal, ethephon,merphos, metoxuron, pentachlorophenol, thidiazuron, tribufos; D5)ethylene inhibitors, such as aviglycine, 1-methylcyclopropene; D6)ethylene releasers, such as ACC, etacelasil, ethephon, glyoxime; D7)gametocides, such as fenridazon, maleic hydrazide; D8) gibberellins,such as gibberellins, gibberellic acid; D9) growth inhibitors, such asabscisic acid, ancymidol, butralin, carbaryl, chlorphonium,chlorpropham, dikegulac, flumetralin, fluoridamid, fosamine, glyphosine,isopyrimol, jasmonic acid, maleic hydrazide, mepiquat, piproctanyl,prohydrojasmon, propham, tiaojiean, 2,3,5-tri-iodobenzoic acid; D10)morphactins, such as chlorfluren, chlorflurenol, dichlorflurenol,flurenol; D11) growth retardants, such as chlormequat, daminozide,flurprimidol, mefluidide, paclobutrazol, tetcyclacis, uniconazole; D12)growth stimulators, such as brassinolide, brassinolide-ethyl, DCPTA,forchlorfenuron, hymexazol, prosuler, triacontanol; D13) unclassifiedplant growth regulators, such as bachmedesh, benzofluor, buminafos,carvone, choline chloride, ciobutide, clofencet, cyanamide, cyclanilide,cycloheximide, cyprosulfamide, epocholeone, ethychlozate, ethylene,fuphenthiourea, furalane, heptopargil, holosulf, inabenfide, karetazan,lead arsenate, methasulfocarb, prohexadione, pydanon, sintofen,triapenthenol, trinexapac.

Plant growth promotors: E1).

Concentrates for Agricultural Compositions

The present invention also relates to a concentrate for an agriculturalcomposition, the concentrate comprising: a) Bacillus amyloliquefaciensFCC1256, and b) an agriculturally suitable formulation medium, e.g.comprising a suitable carrier, a surface-active agent, and optionally abuffer.

Agricultural formulations can be obtained by proper dilution of theconcentrates with a suitable liquid carrier as specified further abovefor the agricultural compositions, in particular an aqueous carrier. Theterm “aqueous carrier” is intended to mean a liquid carrierpredominantly based on water and comprising up to 5% by weight ofnon-water constituent(s). In some embodiments, dilution is by usingwater only.

Typically, dilution is at a concentrate-to-carrier weight ratio of from1:10 to 1:5000, such as from 1:20 to 1:1000.

In embodiments, the concentrate is a formulation selected fromsuspension concentrates (SC or SD), ultra-low volume suspensions (SU),seed treatment suspensions (FS), oil dispersions (OD), pastes (PA), gels(GD), water-dispersible granules (WG), dustable powders (DP),water-dispersible tablets (WT), water-dispersible powders for slurrytreatment (WS) and wettable powders (WP).

Typically, Bacillus amyloliquefaciens FCC1256 is included in suitableformulations in the form of spores thereof, rather than in the form ofvegetative cells. For example, the formulations may include an aliquotof a fermentation broth comprising spores of FCC1256 or a processedfermentation product comprising spores of FCC1256, such as a concentrateof a fermentation broth or a spray-dried fermentation broth.

In some embodiments, the concentrate is in liquid form, and the Bacillusamyloliquefaciens FCC1256 is present in the concentrate at aconcentration of from 1.0×10⁸ CFU/mL to 1.0×10¹⁴ CFU/mL, such as from1.0×10⁹ CFU/mL to 1.0×10¹³ CFU/mL.

In embodiments where the concentrate is in liquid form, the concentratemay be in the form of a formulation selected from suspensionconcentrates (SC or SD), ultra-low volume suspensions (SU), seedtreatment suspensions (FS), oil dispersions (OD), pastes (PA), gels(GD), in particular suspension concentrates (SC) and oil dispersions(OD).

In other embodiments, the concentrate is in solid form, and the Bacillusamyloliquefaciens FCC1256 is present in the concentrate at aconcentration of from 1.0×10⁸ CFU/g to 1.0×10¹⁴ CFU/g, such as from1.0×10⁹ CFU/g to 1.0×10¹³ CFU/g.

In embodiments where the concentrate is in solid form, the concentratemay be in the form of a formulation selected from water-dispersiblegranules (WG), dustable powders (DP), water-dispersible tablets (WT),water-dispersible powders for slurry treatment (WS) and wettable powders(WP), in particular wettable powders (WP) and water-dispersible granules(WG).

In some embodiments, the concentrate comprises iturin and fengycin in arelative weight ratio of in the range of from 1.3:1.0 to 3.0:1.0, suchas from 1.5:1.0 to 2.8:1.0, including all measurements within suchranges.

EXAMPLES

The following Examples have been included to provide guidance to one ofordinary skill in the art for practicing representative embodiments ofthe presently disclosed subject matter. Considering the presentinvention and the general level of skill in the art, those of skill canappreciate that the following Examples are intended to be exemplary onlyand that numerous changes, modifications, and alterations can beemployed without departing from the scope of the presently disclosedsubject matter.

Methods Identification of Iturin, Fengycin and Surfactin

Samples were analyzed by reverse phase LC-DAD using a C18 column with ascreening gradient that allowed the simultaneous detection of all threelipopeptide families. The mobile phases consisted of water andacetonitrile premixed with 0.1% formic acid. Compounds were identifiedbased on their retention times and UV spectra compared with authenticstandards.

Determination of Colony-Forming Units (CFU)

The CFU of a sample (expressed as CFU/mL for liquid samples and as CFU/gfor solid samples) can be determined according to BS EN 15784:2009(“Animal feeding stuffs. Isolation and enumeration of presumptiveBacillus spp.”).

Example 1 Identification of a Bacterial Isolate as a BacillusAmyloliquefaciens Through Sequence Analysis

A plant associated bacterial strain, designated herein as Bacillusamyloliquefaciens FCC1256, was isolated from strawberry plant soil fromAutryville, N.C., USA. The strain of Bacillus amyloliquefaciens FCC1256was deposited on 12 May 2015 under the terms of the Budapest Treaty onthe International Recognition of the Deposit of Microorganisms for thePurposes of Patent Procedure at the American Type Culture Collection(ATCC) in Manassas, Va., USA and bears the Patent Accession No.PTA-122162.

Initially, the whole genome sequence data of Bacillus amyloliquefaciensFCC1256 was mined to identify core phenotypic marker genes that areroutinely used for phylogenetic assignment. Genes that are chosen forthis analysis are both single copy, there is only one copy of the genein the genome, and has a core housekeeping function for the cell. Thesix genes that were chosen from the RAST annotation file were theDNA-directed RNA polymerase beta subunit (rpoB), DNA repair recombinaseA (recA 2), DNA mismatch repair protein S (mutS), glycerol uptakefacilitator protein (glpF), DNA gyrase subunit B (gyrB) and DNAchaperone K (dnaK). For each of the selected genes, representativestrains of genomes for the genus Bacillus (Bacillus subtilis cluster andBacillus cereus cluster) were used for comparison. To that end, FCC1256was identified as a new strain of Bacillus amyloliquefaciens throughsequence analysis of highly conserved 16S rRNA and rpoB genes.

Also included for each gene is an outgroup sequence. These outgroupsequences are mined from genomes of phylogenetically divergent bacteriato provide an indication of the robustness of the phylogeneticcalculations.

Each reference genome was first downloaded from NCBI and annotated inthe RAST annotation pipeline. Each of the annotation tables generatedwas subsequently searched to identify each of the selected housekeepinggenes and the sequences for each gene was then copied and pasted into anew nucleotide alignment in the MEGA 5.2 software program. Once eachgene sequence has been loaded into the alignment all specific genesequences were aligned using the ClustalW alignment algorithm. Aftereach group of genes were aligned the sequences were then trimmed toensure that each sequence had the same number of nucleotides. Followingthe trimming step, the aligned sequences were then used to createphylogenetic trees.

Each phylogenetic tree was replicated 1000 times to assess therobustness of the phylogenetic grouping. For each tree FCC1256 alignsand trees with other Bacillus amyloliquefaciens strains like DSM7,RTI301 and RTI472. These results indicate with high confidence thatFCC1256 is indeed a Bacillus amyloliquefaciens.

The antagonistic effect of FCC1256 against a number of pathogens wasalso determined. Table 1 below provides a summary of the results.

TABLE 1 Antagonistic properties of Bacillus amyloliquefaciens FCC1256against major plant pathogens Anti-Microbial Assays FCC1256 Fungalpathogens: Aspergillus flavus ++ Botrytis cinerea +++ Fusariumgraminearum +++ Fusarium oxysporum ++ Fusarium virguliforme +Phytophthora capsici + Rhizoctonia solani ++ Bacterial pathogens:Erwinia amylovora ++ Xanthomonas euvesicatoria ++ +++ very strongactivity, ++ strong activity, + activity, +− weak activity, − noactivity observed

In addition, experiments were performed to determine the antagonisticactivities of Bacillus amyloliquefaciens FCC1256 in vitro and in variousplants under varying conditions. The experimental results are providedin Examples 3, 5, 6 and 7 below.

The experiments i.a. show the ability of Bacillus amyloliquefaciensFCC1256 to confer protection against or control plant pathogenicinfection as compared to commercially available Serenade® (BayerCropScience, Inc.) that contains as an active ingredient Bacillusvelezensis QST713.

Example 2 Fermentation of Bacillus Amyloliquefaciens FCC1256

Bacillus amyloliquefaciens FCC1256 was first streaked out from aglycerol stock onto a solid 869 media, which contained (in 1 L): 10 gtryptone, 5 g yeast extract, 1 g D-glucose, 0.3 g CaCl₂, and 15 gpowdered agar. After incubating at 30° C. overnight, a single colony waspicked up to inoculate a 250-mL shake flask with 50 mL of mediacontaining (in 1 L): 10 g peptone, 6 g yeast extract, 2 g KCl, and 0.1 gMgSO₄.7H₂O. After autoclaving, the following solutions were added to themedium: 5 mL of 20% glucose, 1 mL of 1M CaCl₂, 1 mL of 0.1 M MnCl₂, and10 μL of 0.1 M FeSO₄. The shake flask was incubated in a shakingincubator at 30° C. and 200 rpm until the OD600 reached 1.0. The cellpellets were collected for the inoculation of fermenters (working volume1.5 L, Bioflo 320, Eppendorf). The fermentation was made at 30° C.,agitation speed was 800 rpm, and the air supply rate was 2 L/min.Samples were taken during the fermentation to monitor cell growth, cellsporulation, and metabolite concentrations. After 168 hours offermentation, most of the Bacillus amyloliquefaciens FCC1256 cells hadsporulated, and the whole culture mixture was harvested.

Bacillus amyloliquefaciens FCC1256 and reference strain QST713 (aBacillus velezensis strain obtained from a commercial Serenade® product)were fermented in the above-mentioned culture medium. Using the OD600measurement, the biomass levels during fermentation were measured. Acomparison of the metabolite profiles for the Bacillus amyloliquefaciensFCC1256 strain and QST713 showed that FCC1256 produced iturin at asignificantly higher rate than QST713 under the same conditions.

Further evaluation of the metabolite concentrates revealed that FCC1256had an iturin:fengycin:surfactin weight ratio at about 3.3:1.4:1,potentially contributing to the antagonistic activities of the strainagainst plant pathogens.

Example 3 Anti-Microbial Properties of Bacillus AmyloliquefaciensFCC1256

Bacillus amyloliquefaciens FCC1256, and QST713 were plated and grown inTSA plates overnight (16-18 h) at 30° C. Following, a single colony wastransferred in medium (growth medium H2O, 914.3 mL; NH₄NO₃, 8 g;Na₂HPO₄, 7.15 g; KH₂PO₄, 6.8 g; yeast extract 0.5 g; adjusted to 7.5with 4 mL 4 M KOH; 45 mL of the growth medium were transferred to a250-mL baffled flasks and autoclaved at 121° C. for 15 min. Afterautoclaving the following solutions were added in each flask: 0.1 MCaCl₂ 5 μL, 0.1M MnCl₂ 5 μL, 0.1M FeSO₄ 35 μL, 0.1M ZnCl₂ 5 μL, 1M MgSO₄100 glucose 50% sol (final conc. 40 g/L) 4 mL) and incubated for 168 hat 30° C. and under continuous agitation (220 rpm). The strains weregrown in 250-mL baffled flasks with GL 45 thread (Duran®, Germany) and50 mL final working volume.

In total, 14 foliar plant pathogens (12 fungi and 2 bacteria) were usedduring this study. Specifications of the pathogens, the growthconditions and media used are shown in Table 2. All foliar pathogensused for this experiment were tested in Potato Dextrose Agar (PDA) solidmedium except Pseudomonas syringae and Xanthomonas axonopodis which weretested in Trypticase soy agar (TSA) solid medium. The incubation time ofeach pathogen before treatment varied from 3 to 5 days. In case ofPseudomonas syringae and Xanthomonas axonopodis where lawn plates areused for testing, an overnight culture on 869 media was prepared whileSeptoria tritici was cultured on Potato Dextrose Broth for 5 days beforelawn preparation.

The activity was assessed by the application of 20 μL of cells. Allapplications were conducted in triplicates for all pathogens using thefermented material non-filtered. Rating timings for fungal pathogensvary by species. Timing should correspond to the colony edge reaching aradius of approximately 25 mm on the control spot plates. For theassessment, a semi-quantitative method was used, cf. the following4-scale system (0-3) where: 0: no antimicrobial effect seen; 1: slighteffect caused but no obvious clearing zone; 2: a clearing zone isobvious; 3: large clearing zone is present. The results are shown inTable 2.

TABLE 2 Anti-microbial properties of FCC1256 against foliar pathogensDays of incubation Incubation before/after Rating Rating Genus andspecies conditions Transfer type treatment QST713 FCC1256 Botrytiscinerea (Bc) Dark, 22° C. Agar plug 0/4 2 2 Stagnospora nodorum (Sn)Dark, 22° C. Agar plug 0/6 2 2 Septoria tritici (St) Dark, 22° C. Agarplug 5/6 2 3 Fusarium oxysporum (Fo) Dark, 22° C. Agar plug 0/4 2 1Fusarium graminearum (Fg) Dark, 22° C. Agar plug 0/4 2 3 Fusariumculmorum (Fc) Dark, 22° C. Agar plug 0/4 2 2 Monilina fructicola (Mf)Dark, 22° C. Agar plug 0/4 2 3 Sclerotinia homeocarpa (Sh) Dark, 22° C.Agar plug 0/3 2 3 Pyrenophora teres (Pt) Dark, 22° C. Agar plug 0/4 2 3Alternaria solani (As) Dark, 22° C. Agar plug 0/6 2 3 Magnaporthe oryzae(Mo) Dark, 22° C. Agar plug 0/6 2 3 Glomerella cingulata (Gc) Dark, 22°C. Agar plug 0/4 2 3 Pseudomonas syringae (Pss) Dark, 30° C. Lawn 1/2 1— Xanthomonas axonopodis (Xa) Dark, 30° C. Lawn 1/2 2 2

Example 4 Phenotypic Traits of Bacillus Amyloliquefaciens FCC1256

In addition to the antagonistic properties, various phenotypic traitswere also measured for Bacillus amyloliquefaciens FCC1256 and the dataare shown below in Table 3. The assays were performed according to theprocedures described in the text below Table 3.

TABLE 3 Phenotypic Assays: phytohormone production, acetoin and indoleacetic acid (IAA), and nutrient cycling of Bacillus amyloliquefaciensFCC1256. Characteristic Assays FCC1256 Acetoin production (MR-VP) +++Chitinase activity + Indole-3-Acetic Acid production − Protease activity++ Phosphate solubilization + +++ very strong, ++ strong, + some, +−weak, − none observed

Acetoin Test. 20 μL of a starter culture in media was transferred to 1mL Methyl Red Voges Proskauer media (Sigma Aldrich 39484). Cultures wereincubated for 2 days at 30° C. 200 rpm. 0.5 mL culture was mixed with0.3 mL 5% alpha-naphthol (Sigma Aldrich N1000) followed by 0.1 mL 40%KOH. Samples were interpreted after 30 min. of incubation. Developmentof a red color indicated acetoin production. Non-inoculated media wasused as a negative control (Sokol et al., 1979, Journal of ClinicalMicrobiology 9: 538-540).

Chitinase activity. 10% wet weight colloidal chitin was added tomodified PVK agar medium (10 g glucose, 0.2 g potassium chloride, 0.5 gammonium sulfate, 0.2 g sodium chloride, 0.1 g magnesium sulfateheptahydrate, 0.5 g yeast extract, 2 mg manganese sulfate, 2 mg ironsulfate and 15 g agar per liter, pH 7, autoclaved). Bacteria were platedon these chitin plates; zones of clearing indicated chitinase activity(N. K. S. Murthy & Bleakley, 2012. “Simplified Method of PreparingColloidal Chitin Used for Screening of Chitinase ProducingMicroorganisms”. The Internet Journal of Microbiology 10(2)).

Indole-3-Acetic Acid. 20 μL of a starter culture in media wastransferred to 1 mL 1/10 869 media supplemented with 0.5 g/L tryptophan(Sigma Aldrich T0254). Cultures were incubated for 4-5 days in the darkat 30° C., 200 rpm. Samples were centrifuged and 0.1 mL supernatant wasmixed with 0.2 mL Salkowski's Reagent (35% perchloric acid, 10 mMFeCl₃). After incubating for 30 min. in the dark, samples resulting inpink color were recorded positive for IAA synthesis. Dilutions of IAA(Sigma Aldrich I5148) were used as a positive comparison; non-inoculatedmedia was used as negative control (Taghavi, et al., 2009, Applied andEnvironmental Microbiology 75: 748-757).

Protease Activity. Bacteria were plated on 869 media supplemented with10% milk. Clearing zones indicated the ability to break down proteinssuggesting protease activity (Sokol et al., 1979, Journal of ClinicalMicrobiology 9: 538-540).

Phosphate Solubilizing Test. Bacteria were plated on Pikovskaya (PVK)agar medium consisting of 10 g glucose, 5 g calcium triphosphate, 0.2 gpotassium chloride, 0.5 g ammonium sulfate, 0.2 g sodium chloride, 0.1 gmagnesium sulfate heptahydrate, 0.5 g yeast extract, 2 mg manganesesulfate, 2 mg iron sulfate and 15 g agar per liter, pH 7, autoclaved.Zones of clearing were indicative of phosphate solubilizing bacteria(Sharma et al., 2011, Journal of Microbiology and Biotechnology Research1: 90-95).

Example 5 Bacillus Amyloliquefaciens FCC1256 Antagonism on Pepper GreyMold (Botrytis Cinerea)

Studies were performed in the greenhouse on pepper to determine theability of FCC1256 to prevent and/or ameliorate the effects of the plantpathogen Pepper Grey Mold (Botrytis cinerea).

Formulations:

Isolated spores of FCC1256 were formulated in 100% spent fermentationbroth (SFB) at a concentration of 1×10⁸ CFU/mL (i.e. a reconstitutedfermentation broth concentrate). A similar FCC1256 formulation wasprepared but with added nutrients (2 g sucrose+1.5 g yeast extract+0.2 gMgSO₄.7H₂O per liter).

Isolated spores of Bacillus amyloliquefaciens RTI472 were similarlyformulated in 100% spent fermentation broth (SFB) at a concentration of1×10⁸ CFU/mL. A similar RTI472 formulation was prepared by with addednutrients (as above for FCC1256).

Serenade® Optimum (Bayer CropScience, Inc.) was applied at a sporeconcentration of 1×10⁸ CFU/mL.

Horizon (Horizon AG-Products) was applied at a rate of 50 g a.i./ha(Tebuconazole).

Treatment Application Method:

A track sprayer was used to inoculate 28 days old pepper plants in agreenhouse with each of the formulations described above. The tracksprayer had a single overhead nozzle (TeeJet SS8001E Flat Fan) at apressure of 276 kPa (40 psi). The nozzle height was 36 cm (14″) abovethe pepper plant leaves. The application volume was 200 L/ha and thenumber of repetitions in the experiment equaled six. The treatmentplants were inoculated a single time along with control plants notreceiving any treatment.

Infection Rate:

One day following treatment application, the test plants were infectedwith Botrytis cinerea at an infection rate of 500,000 conidia/mL.

Three days following infection with Pepper Grey Mold (Botrytis cinerea)the percent of disease control (mean) was evaluated for each of theabove-described formulations.

The results of the experiment are shown in Table 4 below and indicate asuperior control of Pepper Grey Mold (Botrytis cinerea) as compared toRTI472 and Serenade® Optimum when applied at the same rate.

TABLE 4 Results of B. amyloliquefaciens FCC1256 control of Pepper GreyMold (Botrytis cinerea) as compared to Serenade ® Optimum and otherreferences. Treatment (each 1 × 10⁸ CFU/mL) Percent Disease ControlFCC1256 + 100% SFB 98 (±1) FCC1256 + 100% SFB + nutrients 98 (±1)Nutrients 0 RTI472 + 100% SFB 96 (±1) RTI472 + 100% SFB + nutrients 87(±4) Serenade ® Optimum 90 (±2) Horizon 100 (±0)

Example 6 Bacillus Amyloliquefaciens FCC1256 Antagonism on PepperPhytophthora Blight

Studies were performed in the greenhouse on pepper to determine theability of FCC1256 to prevent and/or ameliorate the effects of the plantpathogen Pepper Phytophthora Blight (Phytophthora capsici).

Formulations:

Isolated spores of FCC1256 were formulated in 100% spent fermentationbroth (SFB) at a concentration of 1×10⁸ CFU/mL (i.e. a reconstitutedfermentation broth concentrate). A similar FCC1256 formulation wasprepared but with added nutrients (2 g sucrose+1.5 g yeast extract+0.2 gMgSO₄.7H₂O per liter).

Isolated spores of Bacillus amyloliquefaciens RTI472 were similarlyformulated in 100% spent fermentation broth (SFB) at a concentration of1×10⁸ CFU/mL. A similar RTI472 formulation was prepared by with addednutrients (as above for FCC1256).

Serenade® Soil (Bayer CropScience, Inc.) was applied at a sporeconcentration of 1×10⁸ CFU/mL.

Ridomil Gold (Syngenta, Inc.) was applied at a rate of 0.5 lb a.i./acre(mefenoxam).

Treatment Application Method:

The soil of 28 days old pepper plants was drenched with 10 mL of watercontaining each of the formulations described above. The number ofrepetitions in each experiment equaled six. The treatment plants wereinoculated a single time along with control plants not receiving anytreatment.

Infection Rate:

For each of the studies, on the same day as treatment application, thetest plants were infected with Phytophthora capsici FF157 at a rate of 1mL of 1,000 zoospores/mL.

Eight days following infection of the plants with Pepper PhytophthoraBlight (Phytophthora capsici) the percent of disease control wasevaluated for each of the treatments. The disease severity score was 9.8(0=healthy; 10=dead) at 8 days following infection.

The results showed a statistically superior control of PepperPhytophthora Blight by the FCC1256 as compared to Serenade® Soil whenapplied at the same rate.

TABLE 5 Results of Bacillus amyloliquefaciens FCC1256 control of PepperPhytophthora Blight as compared to Serenade ® Optimum. Treatment PercentDisease Control FCC1256 + 100% SFB 79 (±14) FCC1256 + 100% SFB +nutrients 44 (±18) Nutrients 6 (±6) RTI472 + 100% SFB + nutrients 6 (±6)Ridomil Gold 100 (±0) Serenade ® Soil 30 (±8)

Example 7 Detached Pepper Leaf Disk Assay Comparing the Activity ofBacillus Amyloliquefaciens FCC1256 to Untreated Controls as Well as tothe Commercial Product Serenade® Optimum.

Pepper (Piper capsicum) plants were grown in greenhouse at 20-25° C.with 14-16 hours daylight for 6-7 weeks. From each plant, four fullyexpanded leaves from the middle part were harvested and put in petridishes with moist filter paper. Leaves were sprayed with water(untreated control) or suspension of Serenade® Optimum (positivecontrol) or FCC1256 fermentates (two different batches). Then lids wereput on thee petri dishes and placed for incubation in a growth chamberset at approx. the same growth conditions as the greenhouse.

The following day the leaves were inoculated by spraying with a sporesuspension of Botrytis cinerea, adjusted to 500,000 spores/mL. Then thepetri dishes were put back in the growth chamber and evaluated after 4days, 6 days and 9 days to follow the progression of the disease.

The results are shown in FIG. 1. Column 1 shows the Serenade Optimumreference (application equivalent of 2.8 kg/ha); Column 2 shows theuntreated inoculated control; Column 3 shows the untreated uninoculatedcontrol, Columns 4 and 5 show Bacillus amyloliquefaciens FCC1256 of twodifferent fermentation batches. Rows 1 and 2 show results 4 days postinoculation. Rows 5 and 6 show the same leaves but 6 days postinoculation, and rows 3 and 4 show again the same leaves but 9 days postinoculation.

By visual inspection it is clearly see that the leaves of the untreatedinoculated control (column 2) were highly damaged even after 4 days, andthat the leaves of the untreated uninoculated control (column 3)remained fresh throughout the 9 days experiment. The Serenade® Optimumreference showed some pathogen control after 3 days, by the leaves werehighly damaged after 6 and 9 days. On the other hand, the FCC1256fermentates showed very good pathogen control after 4 and 6 days, andacceptable control after 9 days.

Example 8 (a) Concentrate of Bacillus Amyloliquefaciens FCC1256

A concentrate of FCC1256 was prepared as follow: 20% FCC1256 fermentateconcentrate, 45% water, 30% glycerin (86.5%), 5% Marasperse AG (sodiumlignosulfonate; anionic dispersant) were mixed, pH was adjusted to6.0-7.5 using a HK₂PO₄.H₂O/KH₂PO₄ buffer. The spore content (FCC1256) ofthe final composition was typically in the range of 1.0×10¹⁰ CFU/mL to1.0×10¹² CFU/mL.

(b) Concentrate of Bacillus Amyloliquefaciens FCC1256

A composition of FCC1256 was prepared as follow: 50% FCC1256 fermentateconcentrate, 4.8% water, 40% glycerin (86.5%), 5% Marasperse AG (sodiumlignosulfonate; anionic dispersant), 0.1% antifoam and 0.1% Kelzan(thickener) were mixed, pH was adjusted to 6.0-7.5 using aHK₂PO₄.H₂O/KH₂PO₄ buffer. The spore content (FCC1256) of the finalcomposition was typically in the range of 1.0×10¹⁰ CFU/mL to 1.0×10¹²CFU/mL.

(c) Agricultural Composition of Bacillus Amyloliquefaciens FCC1256

A concentrate of (a) or (b) as specified above was diluted with water ata ratio of 1:12 to produce an agricultural composition. The compositionwas applicable for application as at rate of e.g. 200-600 L/ha.

Example 9 Whole Plant Pepper Assay Comparing the Antifungal Activity ofBacillus Amyloliquefaciens FCC1256 Against Pepper Grey Mold (BotrytisCinerea) to Untreated Controls as Well as to Other BacillusAmyloliquefaciens Strains

Four different Bacillus amyloliquefaciens strains, FCC1256, DSM7, RTI472and RTI301, were fermented under similar conditions (cf. Example 2),except that the fermentations were conducted in a smaller scale using500 mL shake flasks with 60 mL culture. The growing temperature was 28°C., the agitation was 200 rpm and the incubation time was 144 hours. Thefermentations each provided a spore concentration in the order of 1×10⁷CFU/mL.

Pepper plants (Piper capsicum variety Lamuyo F1) were produced in 7×7×7cm plastic pots, using Agrofino 201 soil substrate. They were aged of 6weeks at the time of the test. Watering of the plants was provided everytwo days using fertilization of N:P:K=20:20:20 at 160 ppm. Sixreplicates were used per tested modality, each replicate consisting in 1plant (1 pot).

Each plant was treated inside a laboratory sprayer using a 3-nozzle boomat an estimated volume of 1000 L/ha. Each of the four different Bacillusamyloliquefaciens strains, FCC1256, DSM7, RTI472 and RTI301, were usedat a dose of 20 mL of fermentation broth diluted in 100 mL ofdemineralized water and was mixed with 0.2% vol/vol of an adjuvantmixture. The fermentation medium with the 0.2% vol/vol adjuvant mixtureand without Bacillus strain was also evaluated as a reference.

After application, plants were stored in growth chamber at 20° C., 70%RH and 16 hours photo-period. Inoculation was carried out 24 hours afterfungicide application, by manually spraying inoculum of Botrytis cinereaon treated plants up to run-off. Inoculum concentration was 50,000spores/mL inside PDB medium (strain of Botrytis cinerea FF248). Afterinoculation, plants were stored for 48 hours in a dew chamber at 20° C.(no light) to favor infection. Plants were then place in a growthchamber at 20° C., 70% RH and 16 hours photo-period, up to diseaserating.

Disease assessment was done three days after inoculation, by evaluating% severity on four fully developed leaves per plant. The averaged %severity per pot was then calculated (reported in Table 6), and theefficacy of the tested strains was calculated using Abott formula:

% control A=100*((% severity A−% severity untreated)/% severityuntreated)

TABLE 6 Results of Bacillus amyloliquefaciens FCC1256 control of PepperGrey Mold (Botrytis cinerea) as compared with Bacillus amyloliquefaciensstrains RTI301, DSM7 and RTI472. Strain Percent Disease control RTI30126 (±26) DSM7 72 (±10) RTI472 83 (±11) FCC1256 94 (±6) Fermentationmedium (reference) 3 (±5) Untreated (control) 0 (±0)

REFERENCES

All publications, patent applications, patents, and other referencesmentioned in the specification are herein incorporated by reference intheir entireties.

Although the foregoing subject matter has been described in some detailby way of illustration and example for purposes of clarity ofunderstanding, it will be understood by those skilled in the art thatcertain changes and modifications can be practiced within the scope ofthe appended claims.

1. A method of controlling plant pathogen(s) on a plant, the methodcomprising the step of applying a composition comprising Bacillusamyloliquefaciens FCC1256 deposited as ATCC No. PTA-122162 to the plant,to a part of the plant and/or to a locus at which the plant or plantpart grows or is to be planted.
 2. A method of controlling fungal plantpathogen(s) and/or bacterial plant pathogen(s) on a plant, the methodcomprising the step of applying a composition comprising Bacillusamyloliquefaciens FCC1256 deposited as ATCC No. PTA-122162 toover-ground parts of the plant.
 3. The method of claim 1, wherein thecomposition is in liquid form, and wherein the Bacillusamyloliquefaciens FCC1256 is present in the composition at aconcentration of from 1.0×10⁶ CFU/mL to 1.0×10¹² CFU/mL.
 4. The methodof claim 1, wherein the composition is in the form of a liquidformulation selected from suspensions, suspension concentrates (SC), oildispersions (OD) and foams.
 5. The method of claim 1, wherein thecomposition is in solid form, and wherein the Bacillus amyloliquefaciensFCC1256 is present in the composition at a concentration of from 1.0×10⁶CFU/g to 1.0×10¹² CFU/g.
 6. The method of claim 1, wherein thecomposition is in the form of a solid formulation is selected fromdustable powders (DP), water-dispersible granules (WG) and wettablepowders (WP).
 7. The method of claim 1, wherein the composition isapplied at a rate of from 4.0×10⁹ CFU/ha to 4.0×10¹⁷ CFU/ha.
 8. Themethod of claim 1, wherein the plant pathogen(s) are selected from thegroup consisting of Botrytis cinerea, Botrytis squaomas, Fusariumgraminearum, Fusarium oxysporum, Fusarium viguliforme, Phytophthorainfestans, Phytophthora parasitica, Phytophthora sojae, Phytophthoracapsici, Phytophthora cinnamon, Phytophthora fragariae, Phytophthoraramorum, Phytophthora palmivara, Phytophthora nicotianae, Sclerotiniasclerotiorum, Sclerotinia minor, Sclerotinia homeocarpa, AspergillusFavus, Pseudomonas syringae pv. Tomato, Erwinia amylovara, Rhizoctoniasolani, Xanthomonas euvesicatoria, and any combinations thereof.
 9. Themethod of claim 1 any one of the preceding claims, wherein the plant isselected from pepper, cucumbers, apples, asparagus, berries, bananas,citrus, kiwi, melons, peaches, pears, pineapple, pome fruit,pomegranate, celery, onions, garlic, grapes, leaks, shallots, chives,broccoli, cabbage, cauliflower, cucurbits, tomatoes, potatoes, wheat,rice and soybeans.
 10. The method of claim 1 any one of the precedingclaims, wherein the composition comprises iturin and fengycin in arelative weight ratio of in the range of from 1.3:1.0 to 3.0:1.0. 11.The method of claim 1 any one of the preceding claims, wherein thecomposition further comprises an agriculturally suitable formulationmedium comprising a suitable carrier, a surface-active agent, andoptionally a buffer.
 12. An agricultural composition comprising: a)Bacillus amyloliquefaciens FCC1256 deposited as ATCC No. PTA-122162, andb) an agriculturally suitable formulation medium comprising a suitablecarrier, a surface-active agent, and optionally a buffer.
 13. Thecomposition of claim 12, comprising iturin and fengycin in a relativeweight ratio of in the range of from 1.3:1.0 to 3.0:1.0.
 14. Thecomposition of claim 12, wherein the composition is a formulationselected from suspensions, suspension concentrates (SC), oil dispersions(OD), foams, dustable powders (DP), water-dispersible granules (WG) andwettable powders (WP).
 15. A concentrate for an agriculturalcomposition, the concentrate comprising: a) Bacillus amyloliquefaciensFCC1256 deposited as ATCC No. PTA-122162, and b) an agriculturallysuitable formulation medium comprising a suitable carrier, asurface-active agent, and optionally a buffer.
 16. The concentrate ofclaim 15, which is in form of a formulation selected from suspensionconcentrates (SC or SD), ultra-low volume suspensions (SU), seedtreatment suspensions (FS), oil dispersions (OD), pastes (PA), gels(GD), water-dispersible granules (WG), dustable powders (DP),water-dispersible tablets (WT), water-dispersible powders for slurrytreatment (WS) and wettable powders (WP).
 17. The concentrate of claim15, comprising iturin and fengycin in a relative weight ratio of in therange of from 1.3:1.0 to 3.0:1.0.